Abstract
Introduction: Several studies have demonstrated constitutive activation of the JAK-STAT pathway in MM through dysregulated signaling of cytokines such as IL-6. In addition to its crucial role in promoting the growth, proliferation and survival of myeloma cells, IL-6 is also a potent stimulator of osteoclastogenesis and influences the tumor microenvironment in the bone marrow (BM) of MM patients by promoting an immunosuppressive milieu. Since JAK1 has been shown to be important for IL-6 signaling in MM, studies to assess the effect of JAK1 inhibition alone and in combination with other anti-MM agents were undertaken.
Methods: The human MM cell lines RPMI8226 and U266 were obtained from ATCC and MM1S was kindly supplied by Steven Rosen, MD (Northwestern University, Chicago, IL). BM aspirates were obtained from patients with MM as approved by the Institutional Review Board (Western IRB BIO 001) and informed consent was obtained in accordance with the Declaration of Helsinki. BM mononuclear cells (MCs) were isolated using density-gradient centrifugation with Histopaque-1077 (Sigma-Aldrich, St. Louis, MO). All cells were maintained in RPMI1640 (Omega Scientific, Tarzana, CA) supplemented with 10% fetal bovine serum (FBS), 2mM l-glutamine, 100 IU/mL penicillin and 100 µg/mL streptomycin, in an atmosphere of 5% carbon dioxide at 37◦C. Primary MM BMMCs were cultured in the presence of the JAK1 selective inhibitor INCB052793 plus a panel of anti-MM agents including the alkylating agents cyclophosphamide (CY), melphalan (MEL), and bendamustine (BEN), the proteasome inhibitor carfilzomib (CAR), dexamethasone (DEX) or the immunomodulatory agents lenalidomide (LEN) and pomalidomide (POM). Cells from RPMI8226 or U266 MM cell lines were cultured in the presence of INCB052793 plus CY, MEL, BEN, CAR, DEX, LEN, or POM. After 48 hours, cell viability was assessed using the MTS assay. For the in vivo studies, mice were implanted with a piece of the human MM tumor LAGk-1A. Seven days post-implantation, mice were randomized into treatment groups, and tumor size was measured on a weekly basis. All in vivo studies were approved by the institutional animal care and use committee.
Results: In vitro studies demonstrated that combinations of INCB052793 with a broad spectrum of anti-MM agents synergistically inhibited the viability of BMMCs from MM patients. INCB052793 plus the three alkylating agents or CAR synergistically inhibited the viability of these cells. INCB052793 plus CY or MEL also significantly decreased the viability of the MM1 cell line. In vivo, LAGk-1A-bearing mice had significantly smaller tumors when treated with INCB052793 alone when compared to vehicle control at day 35 post implantation. This was in contrast to mice treated with single agent DEX, LEN or POM. Although the combination of INCB052793 with DEX, LEN or POM did not synergistically inhibit MM cell line growth in vitro, mice receiving the doublets of INCB052793 and DEX, LEN or POM demonstrated an effect on tumor growth that was superior to the doublets of DEX with LEN or POM. Mice receiving the triple combination of INCB052793 + DEX with LEN or POM demonstrated the most significant reduction in tumor growth compared with all other combinations tested. The inhibition of tumor growth with these combinations was observed throughout the study (through day 70) and all combinations were well tolerated. Concomitant with effects on tumor growth, a significant reduction in serum human IgG levels was also observed. In a separate study also using the LAGk-1A model, we evaluated the combination of INCB052793 with CAR or bortezomib (BOR). Combinations of INCB052793 + CAR or BOR were superior at inhibiting tumor growth when compared to single agent INCB052793.
Conclusion: These in vitro and in vivo preclinical studies demonstrate that the combination of the JAK1 inhibitor INCB052793 with a broad spectrum of anti-MM agents are effective, and provide further support for the clinical evaluation of these drug combinations for treating MM patients. Studies to further understand the mechanistic effects of these combinations on MM signaling and the tumor microenvironment are ongoing.
Disclosures
Berenson: Amgen Inc: Consultancy, Honoraria, Research Funding, Speakers Bureau.
The Plant-Derived Compound Resveratrol in Brain Cancer: A Review
